1.1 Field of the Invention
This invention relates to novel biochips that combine integrated circuit elements, electro-optical excitation and detection systems, and molecular receptor probes in a self-contained integrated microdevice.
1.2 Description of Related Art
Much interest has centered on the development of DNA chips based on high density oligonucleotide arrays and fluorescence analysis such as described by Hacia et al. (J. G. Hacia, L. C. Brody, M. S. Chee. S. P. A. Fodor F. S. Collins in Nature Genetics 14, December 1996). This principle has been commercialized in the Affymetrix(copyright) GeneChip(copyright) which was developed to process large amounts of genetic information. GeneChip(copyright) probe arrays are arranged on single chips in the form of tens of thousands of DNA probes that are designed to fluorescence when hybridized to their targets. The light is scanned with laser light and the light intensity stored for later computations (accessed on Jul. 23, 1997 on the affymetrix.com webserver provided by the Affymetrix Corporation, Santa Clara, Calif.).
Unfortunately, the DNA chips, while much like the microprocessor chips that currently run today""s technology, have yet to be successfully developed into integrated systems that conveniently interpret what information can be captured by DNA chips. Thus, an Affymetrix chip that is stated to detect HIV mutations still requires an external scanning and interpretation of the signals that are generated by a DNA-captured nucleic acid.
The detection of biological species in complex systems is important for many biomedical and environmental applications. In particular, there is a strong interest in developing detection techniques and sensors for use in such applications as infectious disease identification, medical diagnostics and therapy, as well as biotechnology and environmental bioremediation. An objective in developing new techniques and sensors is not only to be able to selectively identify target compounds but to be able to assay large numbers of samples. Yet, there remain problems in re-producibly detecting and measuring low levels of biological compounds conveniently, safely and quickly.
A basic interest has been in the development of inexpensive biosensors for environmental and biomedical diagnostics. Biosensors have been investigated, mostly based on DNA probes and on various systems for analysis of oligonucleotide arrays, but there appears to be limited consideration and development of integrated circuit (IC) gene probe-based biosensors on microchips. Existing systems typically employ photomultipliers or 2-dimensional detectors such as charge-coupled device (CCD) systems which require bulky electronic and data conditioning accessories (Affymetrix(copyright) http, 1997; Schena, et al., 1995; Piunno, et al., 1995; Kumar, et al., 1994; Eggars, et al., 1994; and, Graham, et al., 1992).
There are several methods for selectively identifying biological species, including antibody detection and assay as in the well-known Enzyme-linked Immunosuppresent Assays (ELISA) employing molecular hybridization techniques. Generally speaking, it is possible to identify sequence-specific nucleic acid segments, and to design sequences complementary to those segments, thereby creating a specific probe for a target cell, such as different pathogen cells or even mammalian cells that have mutated from their normal counterparts. In principle, one can design complementary sequences to any identified nucleic acid segment. In many instances, unique sequences specific to an organism may be used as probes for a particular organism or cell type. The quantitative phenotypic analysis of yeast deletion mutants, for example, has utilized unique nucleic acid sequence identifiers to analyze deletion strains by hybridization with tagged probes using a high-density parallel array (Shoemaker et al., 1996).
Hybridization involves joining a single strand of nucleic acid with a complementary probe sequence. Hybridization of a nucleic acid probe to nucleic acid sequences such as gene sequences from bacteria, or viral DNA offers a very high degree of accuracy for identifying nucleic acid sequences complementary to that of the probe. Nucleic acid strands tend to be paired to their complements in double-stranded structures. Thus, a single-stranded DNA molecule will seek out its complement in a complex mixture of DNA containing large numbers of other nucleic acid molecules. Hence, nucleic acid probe (e.g., gene probe) detection methods are very specific to DNA sequences. Factors affecting the hybridization or reassociation of two complementary DNA strands include temperature, contact time, salt concentration, the degree of mismatch between the base pairs, and the length and concentration of the target and probe sequences. In perhaps the simplest procedure, hybridization is performed on an immobilized target or a probe molecule attached on a solid surface such as a nitrocellulose or nylon membrane or a glass plate.
Despite significant strides in developing DNA chips, detection and analysis methods have not been well developed to take advantage of the amount of information that such chips can obtain in a short period of time. A common technique for detecting DNA probes involves labeling the probe with radioactive tags and detecting the probe target hybrids by autoradiography. Phosphorous-32 (32P) is the most common radioactive label used because of its high-energy emission and, consequently short exposure time. Radioactive label techniques, however, suffer several disadvantages, such as limited shelf life. For example, 32P has a limited shelf life because it has a 14-day half-life.
Several optical detection systems based on surface-enhanced Raman fluorescence of visible and near-infrared (NIR) dye probe labels have been investigated (Vo-Dinh, et al., 1987 and Isola, et al., 1996) for non-radioactive detection of tagged gene probes. Fluorescence detection is extremely sensitive when the target compounds or labeled systems are appropriately selected. Indeed, a zeptomole (10xe2x88x9221 mole) detection limit has been achieved using fluorescence detection of dyes with laser excitation (Stevenson, et al., 1994). Even so, detection systems are macro compared to the micro world of DNA arrays, as many detection/analysis methods are mere adaptations from other systems. This means that analysis is relatively slow, compared to data accumulation.
There is therefore a distinct need for development of systems that will allow rapid, large-scale and cost effective use of recently developed DNA biochips.
The invention in its broadest aspect comprises an integrated microchip biosensor device. Such a device employs multiple optical sensing elements and microelectronics on a single integrated chip combined with one or more nucleic acid-based bioreceptors designed to detect sequence specific genetic constituents in complex samples. The microchips combine integrated circuit elements, electrooptics, excitation/detection systems and nucleic acid-based receptor probes in a self-contained and integrated microdevice. A basic biochip, for example, may include: (1) an excitation light source; (2) a bioreceptor probe; (3) a sampling element; (4) a detector; and (5) a signal amplification/treatment system.
The integrated circuit biomicrochips of the present invention comprise an integrated circuit that includes an optical transducer and associated optics and circuitry for generating an electrical signal in response to light or other radiation indicative of the presence of a target biological species, particularly a nucleic acid. The chip may also include a support for immobilizing a bioprobe, which is preferably a nucleic acid. In particular embodiments, a target nucleic acid may be tagged or labeled with a substance that emits a detectable signal; for example, luminescence. Alternatively, the bioprobe attached to the immobilized bioprobe may be tagged or labeled with a substance that emits a detectable or altered signal when combined with the target nucleic acid. The tagged or labeled species may be fluorescent, phosphorescent, or otherwise luminescent, or it may emit Raman energy or it may absorb energy.
The highly integrated biosensors of the present invention are advantageous in part because of fabricating multiple optical sensing elements and microelectronics on a single integrated circuit, and further combining the chip in preferred embodiments with a plurality of molecular hybridization probes (Geiger, et al., 1990 and Aubert, et al., 1988). When the probes selectively bind to a targeted species, a signal is generated that is picked up by the chip. The signal may then be processed in several ways, depending on the nature of the signal.
In one aspect, the present invention concerns an integrated system that includes (1) a targeted nucleic acid sequence in combination with a biological probe which is modified to receive light or other radiation of a first frequency and thereby to emit light or other radiation of a different frequency than the first frequency, and (2) to detect the emitted radiation by means of a phototransducer. The target nucleic acid is typically a uniquely characteristic gene sequence of a pathogen such as a fungus, bacteria, or virus, or other distinct nucleic acid species such as may be found in mutant mammalian cells or in individuals with inherited errors of metabolism. The target nucleic acid is modified or labeled to include a tag or label that emits a signal upon exposure to an incident light or other radiation.
The target nucleic acid may be immobilized onto the integrated microchip that also supports a phototransducer and related detection circuitry. Alternatively, a gene probe may be immobilized onto a membrane or filter which is then attached to the microchip or to the detector surface itself, such as the transducer detector described herein. This approach avoids the need to bind the bioreceptor directly to the transducer and thus is attractive for simplifying large-scale production
In one preferred embodiment of the invention, light of a highly directional or focused nature is impinged on a target nucleic that inherently or by virtue of an appropriate tag or label will emit a detectable signal upon irradiation. The irradiation may be provided by a suitable light source, such as a laser beam or a light-emitting diode (LED). With the Raman, fluorescence and phosphorescence detection modes, the incident light is further kept separate from the emitted light using different light paths and/or appropriate optical filters to block the incident light from the detector.
A target nucleic acid sequence is preferably hybridized with a nucleic acid sequence that is selected for that purpose (bioprobe). As stated earlier, the selected bioprobe is immobilized on a suitable substrate, either on the biochip itself or on a membrane type material that is then contacted or attached to the chip surface. The bioprobe may be labeled with a tag that is capable of emitting light or other non-radioactive energy. Upon hybridization with a target nucleic acid sequence, the hybrid product can be irradiated with light of suitable wavelength and will emit a signal in proportion to the amount of target nucleic acid hybridized, see FIG. 20. The labeled bioprobe may comprise a labeled molecular bioreceptor. Known receptors are advantageous to use because of their known ability to selectively bind with the target nucleic acid sequence. In certain particular examples, the bioreceptor itself may exhibit changes in light emission when its cognate is bound.
In certain applications, it may be desirable to increase the amount of biotarget when only trace quantities are present in a sample. The present invention is compatible with polymerase chain reaction (PCR), which is a technique to amplify DNA sequences.